We have developed a powerful technique (representational difference analysis, or RDA) for the comparison of the differences between two complex genomes. By performing RDA on DNA from human tumor cell and the DNA extracted from normal tissue or blood of the same patient, we have derived probes for loci that have undergone genetic alterations including homozygous deletion, point mutation, loss of heterozygosity and amplification in the tumor cells. Moreover, in principle, we can detect rearrangements and foreign genomes (such as viral genomes), if any are present. We propose to continue to apply this methodology to primary and metastatic breast cancers from humans in order to derive probes that detect genetic lesions. Our long term objectives are to relate this information to the prognosis, treatment and etiology of the disease. Underlying this is the belief that robust and quantitative molecular markers are needed to improve clinical service to the patient with breast cancer. We will derive probes that detect gene amplification in breast cancer, and attempt to correlate specific gene amplifications with the course of disease. We will identify the genes that are commonly amplified. Proteins encoded by such genes may offer new therapeutic targets and more sensitive diagnostic markers. We will also categorize the types of sequence (point) mutations that occur in breast cancer, and correlate this information with disease course. This information may be of use in epidemiologic studies, suggesting separate mechanisms underlying cancer incidence in populations separated by age, geography, or culture and ethnic background. At the same time, we will identify probes for regions commonly undergoing genetic rearrangement in breast cancer, and identify pathogenic genomes, if any are present in breast cancer cells.